Advances in recombinant DNA technology and genetic engineering have provided means for producing in bacteria specific proteins of commercial and economic importance. In many instances, bacteria are unable to consistently provide the post-translational modifications such as proper protein folding, glycosylation, protein processing (e.g. enzymatic cleavage) and the like required for functional eucaryotic protein production. Indeed, studies in eucaryotic cells of such post-translational modifications as glycosylation indicate that the specific post-translational modification may even vary depending upon the eucaryotic cell type doing the modifying. This host cell dependent variability is significant as differences in, for example, protein glycosylation, may result in production of antigenically and/or functionally different proteins. It is, therefore, desirable to develop eucaryotic cell systems (e.g. eucaryotic host-vector systems) which can provide the desired and/or required post-translational modifications and thus become efficient factories for functional and/or antigenically homologous protein production.
A key element in the genetic engineering of both procaryotic and eucaryotic cells to effect the production of a desired protein is the development of defined vectors and host-vector systems. "Vector(s)" or "vector system(s)" are herein defined as nucleic acid molecules capable of introducing a heterologous nucleic acid into a host cell. "Heterologous" when applied to a nucleic acid (e.g. DNA and/or RNA) is herein understood to mean a nucleic acid sequence wherein all or a portion of the sequence or its complement are not normally present in the vector employed to introduce the heterologous nucleic acid into a host cell. A "cloning vector" is herein understood to mean a vector which permits the replication of heterologous nucleic acid within a host cell. An "expression vector" is herein understood to mean a vector which permits the production, in a host cell, of a protein or fragment thereof encoded in the heterologous nucleic acid sequence. The term "host-vector system" is herein understood to mean a host cell capable of accepting and allowing the replication and expression of genetic information carried by a vector.
A number of eucaryotic vector systems have been developed, all are viral vectors, all replicate through a DNA intermediate, and all have some limitation which renders them each less than ideal for expression and/or cloning of heterologous nucleic acid(s). These limitations include high frequencies of spontaneous mutations, Ashman and Davidson (1984); Calos et al. (1983), low levels of expression, Rigby (1983), limited host range and decreased efficiency of heterologous nucleic acid expression due to nuclear regulatory mechanisms such as chromatin structure, transcriptional regulation, polyadenylation, RNA processing and RNA transport to the cytoplasm. Although the poxvirus vector system bypasses all the limitations of nuclear regulation, this system is tedious to work with using currently available methodologies. Thus, while a number of vectors have been developed to replicate and express heterologous nucleic acid in eucaryotic host cells, there is a continued need for eucaryotic vectors able to permit rapid and efficient heterologous nucleic acid expression in a wide range of host cells. Employment of a ssRNA virus genome as a vector would certainly facilitate efficient and rapid expression of heterologous nucleic acid (e.g. RNA) that otherwise may be expressed poorly due to nuclear regulatory mechanisms.
The present invention provides vectors comprising RNA molecules derived from alphavirus genomes and, specifically, from alphavirus defective interfering (DI) RNA. The alphavirus genus of the Togaviradae family includes more than 20 distinct viruses, Schlesinger (1980), the genomes of which comprise a single plus (+) strand RNA molecule which, unlike the current viral genomes employed as eucaryotic vectors, replicates soley through an RNA intermediate. Alphavirus gene expression and viral replication are independent of the host cell nucleus. Additionally, all alphaviruses studied to date have the ability to replicate in a wide range of vertebrates including both avian and mammalian hosts. Sindbis virus, one well-studied member of the alphavirus group, for example, infects all warm-blooded animal (avian and mammalian) cells tested and, in addition, has been shown to infect viper (reptilian) cells, Clark et al. (1973), as well as a large number of mosquito speceis, a fowl mite (Bdellonyssus bursa) and Drosophila melanogaster, Bras-Herring (1975) (1976). Thus, vectors derived from alphaviruses would offer a wide latitude in the choice of host cell consistent with synthesizing a particular product (e.g. protein) of interest. To date, there have been no reports nor teaching of how to construct a vector comprising an alphavirus genomic RNA molecule having inserted therein heterologous RNA.
Furthermore, like many other viruses, alphaviruses exhibit another interesting feature. Specifically, when passaged at high multiplicity in cultured cells, alphaviruses accumulate deletion mutants characterized by their ability to interfere with the replication of standard virus. These mutants are defined as defective interfering or DI particles. Perrault (1981). Depending upon the cells and the conditions of passaging, the first detectable DI particles appear after about three to five high multiplicity passages. The DI RNA contained within these DI particles is about one half the size of standard or virion RNA. Guild and Stollar (1977); Stark and Kennedy (1978). These molecules soon disappear on subsequent passaging and are replaced by molecules one-fourth to one-fifth the size of the original viral genome. In addition to the variable size of the DI RNA's generated by repeated high multiplicity infection of cultured cells by standard virus, the alphavirus DI genomes have been demonstrated to contain a heterogeneous and complex nucleic acid structure. Pettersson (1981). Notwithstanding the heterogeneous size and genome complexity of alphavirus DI RNA's, the biologic life cycle of the DI RNA's suggests that all DI virus genomes should contain the necessary sequences for DI RNA replication and packaging in the presence of helper virus (e.g. coinfection with standard virus). The sequences necessary for either DI or standard virus RNA replication and packaging, however, have not yet been elucidated.
Accordingly, it is an object of the present invention to provide alphavirus DI RNA sequences which permit alphavirus DI RNA replication and packaging in the presence of helper virus.
It is another object of the present invention to provide a vector comprising alphavirus DI RNA having inserted therein heterologous RNA.
It is still another object of the present invention to provide genetically altered alphavirus particles and/or cells comprising alphavirus DI RNA having inserted therein heterologous RNA.
It is yet a further object of the present invention to provide methods and compositions which permit the packaging of heterologous RNA into alphavirus virions.
These and other objects of the invention will be more fully apparent from the following general and detailed description of the invention.